Substance extracted from growth medium of rhizopus nigricans and method of obtaining the same



Patented Aug. 9, 1949 SUBSTANCE ex ra-norm) FROM "GROWTH M'EBIFUM ornmzorns NIGR/ICANS :AND Mentor or OBTAINING THE SAME {mm c. Keresztesy,West'field, and {Edward Rickes, aahwa N. J., assignors to Merck & '00,Inc stairway, N. J., a corporation of New Jersey N Drawing. Applicatio-May 19, 1944, serial No. 536,434

follows xii. 2, 575 2, 149 2, 525 Emma 1s. 6 11(7 12 ml. 2, 650 2,700em-x 14.1 10. 8 iii. 3, 650 3, 500 3, 2'2 5 -r:m 1o 4. s 2.44 4.4

Rhiz'opterinis further characterized by high ac tivity in promotinggrowth of S. Zactzsfalthough 'rlativelyiriactive in promoting growthofLfic'usei. (fine gaiinma "of "rhizopteri'n has substantially the samepotency for S. 'Z'actz's "as about 56 gamma of standard @0110 acidconcentrate, although the sam'e amount of rhizopterin is less activethan about--0.-0004'=gamma or this folic acid standard forL. casei.*Rhizoptrin is but slightly soluble in water, insoluble inorganicsolveiits, soluble in alkali solutions, and when heated, darkensindicating decomposition without melting at -310- 330C.

This new chemical compound, rhizopterin, is obtained according to oneprocess within the scope of this invention'from a charcoal adsorbateprepared by treating a fumaric acidiermentation liquor with activatedcharcoal. The fumaric acid fermentation liquor used for this purpose isatypical growth medium wherein an organism, for-instanceRhizopus-nigricans, is propagated to produce fumaric acid and'theadsorbate is obtained by mixing activated charcoal, for instance NoriteA, with the liquor, filtering and Washing.

The following examples illustrate methods of carrying out the presentinvention, but it is to be understood that these examples are given byway of illustration and not of limitation.

Example 1 I About 180 pounds of moist charcoal adsorbate equivalent 'toabout pounds of dry material, obtained as above described, are eluted--f or about an hour with approximately lQgallons-of-ethanol (30% byvolume) containing sodium hydroxide (about 0.25% by Weight). I he eluateis filtered, acidified with dilute sulfuric acid to about pH 2:3, andmixed with 4 approximately a iiiiogi am of activated charcoal. After anhour, time rina ture is filtered and the residu'e is washed with Water.About '2 kilograms dry 'weigh'tu or this residue are eluted :for a'nhour with a solventconsisting -o'f I5 'gallonsoi ethanol ..33=2i galionsof water and about 3100 "cc. "of sodiuin liy droxidesolution (30% byweight). 'The mixture is then filtered, the filtrate neutralizedaiidcoircentrated to a total volume of about 12 Iitrs. The concentrated:solution is carefully a'cidifietl to aboutipH l causing formation ofaprecipitate which -'is removed, 'ipreferably by ce'ritrifugatidn, washedwith ethanol, water, and acetone, and driedin vacuo. About -150gms.'Of'UI'i'iSGOHCBH- trate are; suspended in approximately 1500 cc. .of

' ammonium hydroxidesolution (6-7 normal) and the mixture is stirred forseveral hours after which insoluble r natte1' is removed preferably --bycentrifugation. 7 The clarified supernatant liquo r isthen mixedwithapproximatly l0 vo1umes*of absolute ethanol and the mixture is againcentrifiiged after which "insoluble pretend is removed."The'clea'rsolutioh'is "acidified with acetic acid to a 'pH of 4i5-5.0arid again precipitated in'a'tter is removedb centralisation. Theclarifie'dsoliitlon ischroniatographed batch' wiseii'pon aluminum oxide;and after washing with" alcohol thealur'ninumoxide is elutedwitha'solvent'c'o'n- Sitifig of five parts methanol, one part concentratedammonia water and sufiic'ient -distilled water to make f'a total 'volumeor To parts. The eliiaitioh "is conducted 'batch tvis'e using portionsof solven't succss'iv'ely 1111611 13116 same "aluminum oxide alnd thefractions exhibiting mest pronounced microbiologicalactivity are seletedfor further operations. Theselected eluate 'fra'ctions are combined andconcentrated in vacuo (40%5" 0:) to a total volume of approximately 120120,. About cc. of concentrated ammonia water are added to thisconcentrate together with two liters of absolute ethanol. If aprecipitate forms it is removed before further operations. The solutionis then acidified with glacial acetic acid to pH 4.5-5 and anyprecipitate formed upon standing is removed. The clarified solution ischromatographed upon aluminum oxide followed by washing with 500 cc. ofethanol (95%), followed by methanol (50%) The aluminum oxide is theneluted as before using a solvent consisting of methanol, water, andconcentrated ammonium hydroxide, the concentrate of ammonium hydroxidemay be 1% of the above and other bases can be used. Againmicrobiological assays are used to select potent eluate fractions. Thesefractions are concentrated in vacuum at a temperature of 3040 C. to avolume of approximately 20 cc. A yellow semi-crystalline materialseparates which is removed by filtration, washed with water and alcoholand dried in vacuum. The product is purified by forming a solution ofsame in ammonium hydroxide, clarifying the solution by centrifugationand adding absolute ethyl alcohol to cause separation of the ammoniumsalt of rhizopterin as aggregates of fine yellow needles. The ammoniumsalt, when dissolved in water and the solution carefully acidified withacetic acid to pH 6 yields buff rhombohedron-like micro crystals ofrhizopterin which, if desired, can be further purified by repeating thecrystallization procedure above described. The product possesses theproperties set forth above in the definition of the term rhizopterin.

Example 2 A charcoal adsorbate from fumaric acid fermentaticn liquor iseluted with alcoholic sodium hydroxide solution and the eluate isacidified, treated with activated charcoal, the charcoal removed, washedand eluted with a solvent consisting of ethanol, water and sodiumhydroxide as in 150 gallons of this eluate are diluted with water toapproximately the foregoing example.

ed in about 1500 cc. of hydrochloric acid /2 normal) and insolublematter is removed by filtration or centrifugation. About 10 volumes ofethanol are added to the filtrate and the pH is adjusted to 3-4.5 byaddition of dilute sodium hydroxide solution. Insoluble material isremoved by filtration and the solution is chromatographed upon aluminumoxide, then eluted with methanol (50%) containing 10% ammoniumhydroxide. Eluate fractions are selected as in the foregoing example,combined, and rechromatographed after which again active eluatefractions are selected and rechromatographed again. The selectedfractions are combined and concentrated in vacuo at 30-40 C. to a totalvolume of approximately 20 cc. Yellowish micro crystals of rhizopterinseparate and are removed by filtration. This is purified by conversionto the ammonium salt which, if desired, is reconverted to rhizopterin bythe procedure set forth in Example 1.

Salts of rhizopterin other than the ammonium salt above described can beprepared by treating rhizopterin solutions with weak organic bases.

Modifications may be made in carrying out the present invention withoutdeparting from the spirit and scope thereof and the invention is to belimited only by the appended claims.

What is claimed is:

1. The process for obtaining rhizopterin that comprises eluting, withalkaline aqueous ethanol, a charcoal adsorbate of a fumaric acidfermentation liquor obtained from the growth medium of Rhizopusnigricans, acidifying the eluate and mixing with activated charcoal,separating and eluting the charcoal with alkaline aqueous ethanol,acidifying this eluate and mixing with fullers earth, separating andeluting the fullers earth with ammoniacal aqueous ethanol, concentratingthis eluate to cause separation of crude rhizopterin as a precipitate,separating and drying this precipitate, dissolving the precipitate inaqueous hydrochloric acid, adding alcohol, then alkali to pH 34.5,removing separated impurities, chromatographing the solution on aluminumoxide, eluting the aluminum oxide with ammoniacal aqueous methanol,evaporating the eluate to obtain rhizopterin.

2. A substance selected from the class consisting of rhizopterin and theammonium salt of rhizopterin, the rhizopterin being obtained by eluting,with alkaline aqueous ethanol, a charcoal adsorbate of a fumaric acidfermentation liquor obtained from the growth medium of Rhizopusnzgricans, acidifying the eluate and mixing with activated charcoal,separating and eluting the charcoal with alkaline aqueous ethanol,acidifying this eluate and mixing with fullers earth, separating andeluting the fullers earth with ammoniacal aqueous ethanol, concentratingthis eluate to cause separation of crude rhizopterin as a precipitate,separating and drying this precipitate, dissolving the precipitate inaqueous hydrochloric acid, adding alcohol, then alkali to pH 3-4.5,removing separated impurities, chromatographing the solution on aluminumoxide, eluting the aluminum oxide with ammoniacal aqueous methanol,evaporating the eluate to obtain rhizopterin, said rhizopterin having anapproximate carbon-hydrogen-nitrogen content to 53% carbon, 4.0%hydrogen, and 25% nitrogen, the balance being oxygen, and being furthercharacterized by being but slightly soluble in water, insoluble inorganic solvents,

soluble in alkali solutions, and, when heated, darkening indicatingdecomposition without melting at 310-330 C.

JOHN C. KERESZTESY. EDWARD RICKES.

REFERENCES CITED The following references are of record in the file ofthis patent:

Foster et al., J. Am. Chem. 800., vol. 61, pp. 127-135 (January 1939).

Chem. Abstrs. 3410, by Foster (1939), vol. 33.

